Monocytes Derived From Human Pluripotent Stem Cells Engineered for Detection of Pyrogens

Pyrogens, including endotoxins and non-endotoxins, are key factors that impact the safety of parenterally administered drugs as contaminants. Currently, traditional pyrogen detection methods primarily rely on the Rabbit Pyrogen Test (RPT) and the Limulus Amebocyte Lysate (LAL) assay, both of which are derived from animal-based systems. However, both methods possess certain limitations. The RPT demonstrates lower reproducibility and a higher false-positive rate compared to the LAL assay; in contrast, the LAL test is limited to detecting endotoxins from Gram-negative organisms. The Monocyte Activation Test (MAT), which is based on monocytes, has emerged as the most promising alternative to traditional pyrogen detection methods. However, the MAT is operationally complex, time-consuming, and exhibits significant individual variability; it typically requires a substantial volume of human blood, thereby considerably limiting its practical application. To overcome these limitations, we developed a rapid pyrogen detection method based on monocytes derived from human embryonic stem cells (hESCs), integrated with a luciferase reporter system. By leveraging the stability and pluripotency of hESCs, we can obtain numerous hESC-derived monocytes (hESC-Mono) through directed in vitro differentiation. These hESC-Mono exhibit batch-to-batch consistency and closely resemble peripheral blood monocytes in functionality. We have demonstrated that hESC-Mono possess sensitive reporting capabilities for at least three types of pyrogens: lipopolysaccharide (LPS), a major component of the cell wall of Gram-negative bacteria; lipoteichoic acid (LTA), a key constituent of the cell wall of Gram-positive bacteria; and fungal Zymosan. Furthermore, we have confirmed that hESC-Mono maintain stable expression of Toll-like receptors, which are among the primary determinants of the reliable detection of pyrogens. Consequently, we have successfully developed and validated a novel pyrogen detection method based on the integration of an in vitro hPSC-directed differentiation system with a luciferase-based reporter assay, thereby offering a promising alternative to traditional pyrogen detection methods.